The Studies On Bio-toxicity Of Ethidium Bromide And Cytoskeleton Of Mesenchymal Stem Cells

Atomic force microscope (AFM) is one of the most important tools and has been extensively used in molecular biology and cellular biology studying. AFM possesses many advantages in life sciences studies, such as the samples need not complex pretreatment and can image in near-physiological condition and obtain various physical data of samples. In addition, AFM is a perfect tool in damnification studying of DNA, chromosome and cell induced by Ethidium bromide (EB), and also ideal tool in cytoskeleton of mesenchymal stem cells (MSCs) studying and cell mitosis visualizing.In this paper, we studied the bio-toxicity of EB on DNA, chromosome and embryo stem cells (ESCs) by AFM. And meanwhile, we also studied the effects of trypsinization treatment on chromosome surface structures during chromosomal preparation, the correlation between the cytoskeleton and ultra structures of MSCs and its function, and the process of Chinese hamster ovary (CHO) Cells Mitosis.The results indicate that (1) the morphological structures of DNA, chromosome and ESCs are obviously deformed by EB. The average height of DNA decreased, and the broken DNA induced by EB is also observed. The damaged location of chromosome is centromere, which is damaged severer than chromosomal arms, and addition of EB into media of cultured human blood cells induced chromosomal breakage in dose-dependent fashion. And as far as ESCs are concerned, the morphological structures have also been deformed distinctly by EB, cellular membrane surface destroyed and hole and pore observed. And similarly, addition of EB into media of cultured ESCs induced its deformation in dose-dependent fashion, the damnification degree of ESCs decreased with the dose of EB reduced. (2) The appropriate digested time of trypsinization is about 10 s, and the morphological structures of chromosome damaged severely even the false structures of "three-strand" observed when the digested time exceed 30 s. (3) AFM possesses many unique advantages in cytoskeleton studying. We studied the particular morphological structures of MSCs such as branch-like, drumstick-like and reticular cytoskeleton and palpus-like and fish cauda-like pseudopodium and so on, which probable correlated with its function tightly.In brief, AFM possesses many advantages over other high-resolution microscopy such as electron microscope (SEM, TEM) and scanning tunneling microscope (STM). The ultra structural deformation of DNA, chromosome, ESCs induced by EB, cytoskeleton and the tracking observation of cellular mitosis have all been visualized by AFM, and we are able to divide metaphase and anaphase into sub-phases: metaphase I , II and III, and anaphase I and II according to chromosomal morphological structures.Our results provide the direct proof for the bio-toxicity of EB, and it is helpful for further understanding its carcinogenic action. The examination of chromosomes treated with trypsin is useful for searching more appropriate chromosomal preparation condition, which is important for AFM imaging. In addition, cytoskeleton of MSCs have been visualized by AFM, which can help us to further understand the correlation between the cytoskeleton and ultra structures of MSCs and its function, and can provide some valuable data for its clinic application.